Targeting protein-diagnostic/therapeutic agent conjugates having Schiff base linkages

ABSTRACT

Targeting protein-diagnostic/therapeutic agent conjugates joined by stabilized Schiff base linkages are disclosed. Schiff base linkage of targeting protein and agent is accomplished without exposure of the targeting protein to harsh oxidizing or reducing conditions. The cleavable, heterobifunctional linkers that are described provide certain advantages relating to in vivo administration of targeting protein conjugates, including controlled release of active agent at a target site.

This application is a divisional of application Ser. No. 08/332,045,filed Nov. 1, 1994 now U.S. Pat. No. 5,633,351.

TECHNICAL FIELD

The claimed invention relates to targetingprotein-diagnostic/therapeutic agent conjugates that are joined by animproved Schiff base linkage that provides advantageous properties forin vivo imaging and therapy.

BACKGROUND OF THE INVENTION

A Schiff base is an imine condensation product of an aldehyde and aprimary amine. Formation of a Schiff base may be illustrated by thefollowing reaction: ##STR1## Where R and/or R' are aliphaticsubstituents, the C═N (imine) bond of a Schiff base is known to be veryunstable. Typically, the C═N bond is stabilized by reduction with sodiumborohydride or sodium cyanoborohydride, as represented by the followingreaction: ##STR2## Stabilization of the C═N bond may also be achievedthrough the attachment of an aryl(s) to the imine carbon or nitrogen, orif a hydroxyl or second nitrogen is bonded to the imine nitrogen.

Schiff base linkages have been used for conjugation of glycoproteins ingeneral, and for conjugation of immunoglobulins in particular. In atypical reaction scheme, oligosaccharide moieties present on animmunoglobulin molecule are oxidized to form one or more aldehydegroups. The resultant immunoglobulin aldehyde(s) is reacted with aprimary amine to form a Schiff base, which is then stabilized byreduction.

A prototypical Schiff base conjugation procedure (as described above)suffers from numerous disadvantages. First, the immunoglobulin(glycoprotein) molecule is subjected to very harsh oxidizing conditionsin order to generate free aldehyde groups. This harsh oxidation mayresult in impairment of the biological activity of the immunoglobulinmolecule. Second, stabilization of the Schiff base conjugate isaccomplished through exposure of the conjugate to a harsh reducingagent, which also may adversely affect the biological function of theimmunoglobulin moiety. Third, the number of substituents that may beconjugated by Schiff base linkage to immunoglobulin aldehyde groups islimited by the number of carbohydrate moieties present on a particularimmunoglobulin molecule. For instance, the amount of carbohydratepresent on an immunoglobulin molecule may vary between 2-3% for IgG and9-12% for IgM, IgD and IgE (I. M. Roitt et al, "Immunology", GowerMedical Publishing Ltd., 1985, p. 5.2).

Alternatively, an aldehyde generated on an immunoglobulin molecule maybe reacted with a hydrazide to form a hydrazone, according to thefollowing reaction scheme: ##STR3## Hydrazones are more stable thanSchiff bases formed by the reaction of an aldehyde and a primary amine,and thus do not require reduction after formation of the linking bond.However, this reaction scheme suffers from several disadvantages: (1)the immunoglobulin must still be oxidized to generate free aldehydegroups; and (2) the degree of conjugation is limited by the number ofcarbohydrate moieties present on the immunoglobulin molecule.

SUMMARY OF THE INVENTION

The present invention provides targeting protein conjugates covalentlybonded to one or more diagnostic or therapeutic agents through improvedSchiff base linkages. Oxidation/reduction of the targeting proteincomponent is eliminated; a variety of targeting protein substituents maybe used to produce a Schiff base-linked conjugate.

Targeting protein--diagnostic/therapeutic agent conjugates joined by anaromatic, heterobifunctional Schiff base linker are also disclosed. Inone embodiment, the Schiff base linkage is formed between the targetingprotein and the linker. In a second embodiment, the Schiff base linkageis formed between the agent and the linker. The latter linker mayfurther provide enhanced target cell retention of the conjugate.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to set forthdefinitions of certain terms to be used within the disclosure.

Schiff base linkage: A chemical bond represented by R--CH═NH--R'.

Stabilized Schiff base linkage: A chemical bond represented byR--CH═NNHCOR' (hydrazone).

Conventional Schiff base linkage: A Schiff base linkage formed byreaction of an aldehyde or ketone group present on a targeting proteinwith a nucleophilic primary amine or hydrazide present on a diagnosticor therapeutic agent.

Unique Schiff base linkage: A Schiff base linkage formed by reaction ofa primary amine or hydrazide present on a targeting protein with analdehyde or ketone present on a diagnostic or therapeutic agent.

Targeting protein: A protein or peptide that binds to a definedpopulation of cells. The targeting protein or peptide may bind areceptor, an enzymatic substrate, an antigenic determinant, or otherbinding site present on the target cell population. Hereinafter, theterm "targeting protein" will be inclusive of both targeting proteinsand targeting peptides.

Conjugate: A two-component hybrid molecule wherein the components arejoined by a covalent chemical linkage.

Targeting protein conjugate: A conjugate wherein one component isantibody (i.e., an immunoconjugate) or, more generally, a targetingprotein. Typically, the second component of a targeting proteinconjugate is a therapeutic agent (i.e., a drug, a toxin or aradionuclide) or a diagnostic agent (i.e., a radionuclide).

A first aspect of the present invention describes a conjugate of atargeting protein and a diagnostic or therapeutic agent covalentlyjoined through one or more stabilized Schiff base linkages.

In a first aspect of the claimed invention, a targeting proteinconjugate is joined through a stabilized unique Schiff base linkage, asrepresented below: ##STR4## wherein "Tp" is a targeting protein (such asa peptide, polypeptide, glycoprotein, carbohydrate-free protein,targeting protein, carrier protein or chelator);

"L1" and "L2" are heterobifunctional linkers having a hydrazide oraldehyde/ketone active group at one end of the linker;

"n" and "n'" are 0 or 1;

"R" is H; an alkyl, aryl, or alicyclic substituent; and

"agent" is a diagnostic or therapeutic agent useful for in vivoapplications, or a chelating agent capable of binding small diagnosticor therapeutic molecules.

Preferred targeting proteins useful within the present invention includeantibody and antibody fragments; peptides, such as bombesin,gastrin-releasing peptide, RGD peptide, substance P, neuromedin-B,neuromedin-C, and metenkephalin; and hormones, such as EGF, α- andβ-TGF, estradiol, neurotensin, melanocyte stimulating hormone, folliclestimulating hormone, luteinizing hormone, and human growth hormone.Biotin, avidin, proteins corresponding to known cell surface receptors(including low density lipoproteins, transferrin, insulin and CD₄),fibrinolytic enzymes, and biological response modifiers (includinginterleukin, interferon, erythropoietin and colony-stimulating factor)are also preferred targeting proteins. Analogs of the above-listedtargeting proteins that retain the capacity to bind to a defined targetcell population may also be used within the claimed invention. Inaddition, synthetic targeting proteins and peptides may be designed.

Exemplary cytotoxic agents include toxins and drugs. Several of thenative toxins useful within the present invention consist of an A and aB chain. The A chain is the cytotoxic portion and the B chain is thereceptor-binding portion of the intact toxin molecule (holotoxin).Because toxin B chain may mediate non-target cell binding, it is oftenadvantageous to conjugate only the toxin A chain to a targeting protein.However, while elimination of the toxin B chain decreases non-specificcytotoxicity, it also generally leads to decreased potency of the toxinA chain-targeting protein conjugate, as compared to the correspondingholotoxin-targeting protein conjugate.

One possible explanation for the decreased potency of A chain-targetingprotein conjugates is that B chain is required for translocation of theA chain across endosomic membranes into the target cell cytoplasm. Inthe absence of translocation, the targeting protein conjugate remains inthe interior of an endosome, and is ultimately transported to alysosome. Within the lysosome, the targeting protein conjugate isdegraded, and thus the A chain cytotoxic agent fails to reach itscytoplasmic target site. The decreased potency associated with toxin Achain-targeting protein conjugates also accompanies the use of ribosomalinactivating protein-targeting protein conjugates. Ribosomalinactivating proteins (RIPs) are naturally occurring protein synthesisinhibitors that lack translocating and cell-binding ability.

Within the present invention, preferred toxins include holotoxins, suchas abrin, ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin,pertussis toxin and Shiga toxin; and A chain or "A chain-like"molecules, such as ricin A chain, abrin A chain, modeccin A chain, theenzymatic portion of Pseudomonas exotoxin A, Diphtheria toxin A chain,the enzymatic portion of pertussis toxin, the enzymatic portion of Shigatoxin, gelonin, pokeweed antiviral protein, saporin, tritin, barleytoxin and snake venom peptides.

Exemplary drugs include daunomycin, adriamycin, vinblastine,doxorubicin, bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine,cytarabine, cyclophosphamide and similar conventional chemotherapeuticsas described in Cancer: Principles and Practice of Oncology, 2d ed., V.T. DeVita, Jr., S. Hellman, S. A. Rosenberg, J. B. Lippincott Co.,Philadelphia, Pa., 1985, Chapter 14. Yet another preferred drug withinthe present invention belongs to the trichothecene family, withverrucarin A particularly preferred. Experimental drugs may also besuitable for use within the present invention (see. e.g., NCIInvestigational Drugs, Pharmaceutical Data 1987, NIH Publication No.88-2141, Revised November 1987).

Conjugates of targeting proteins and cytotoxic agents joined throughnon-stabilized unique Schiff base linkages have been described (Sela etal., U.S. Pat. Nos. 4,093,607 and 4,263,279). Such unique Schiff baseconjugates may be represented by the following formula:

    Protein-NH═CH-cytotoxic agent

which upon reduction yields:

    Protein-NH--CH.sub.2 -cytotoxic agent.

A stabilized unique Schiff base-linked targeting protein conjugate ofthe present invention, as described above, provides certain advantagesas compared to previously described non-stabilized, reduced uniqueSchiff base conjugates:

(1) Various substituents of the targeting protein may be used to producea stabilized unique Schiff base-linked conjugate of the claimedinvention. For instance, a native disulfide bond within the targetingprotein may be used to generate a free sulfhydryl, which in turn reactswith a maleimide end of a heterobifunctional linker having a hydrazideor aldehyde group present at the other end of the linker molecule. Ifthe targeting protein does not possess a native disulfide bond, lysineresidues may be used to introduce free sulfhydryl groups into thetargeting protein through reaction with iminothiolane. In yet anotherembodiment of the invention, targeting protein carboxylic acid groupsmay be reacted with hydrazine to form a targeting protein hydrazide.Thus, either sulfhydryls, lysines or carboxylic acid groups of atargeting protein may be used for production of unique Schiffbase-linked targeting protein conjugates described herein.

In contrast, a non-stabilized unique Schiff base linkage according toSela et al. requires binding of periodate-oxidized drug to free aminogroups of protein to form a non-stabilized imine linkage.

(2) Formation of a stabilized unique Schiff base (hydrazone) linkageaccording to the present invention does not require reduction of theresultant conjugate, for stabilization. The non-stabilized unique Schiffbase (imine) conjugate schematically represented above must be reducedwith sodium borohydride or sodium cyanoborohydride for stabilization ofthe imine bond. Reduction of the imine bond to an amine bond makes thelinkage non-cleavable under biologic conditions. In addition toreduction of the imine bond to an amine bond, exposure to borohydride orcyanoborohydride may also reduce disulfide bonds and amide linkages, andproduce other as yet unidentified deleterious effects on the protein andcytotoxic agent components of the conjugate.

In one embodiment of this aspect of the invention, lysine groups of atargeting protein (either glycoprotein or non-glycoprotein), are treatedwith a reagent (for instance, iminothiolane) that adds free sulfhydrylgroups to the targeting protein. The sulfhydryl-derivatized targetingprotein (Tp-SH) is then reacted with a heterobifunctional linker havinga maleimide reactive group.

An exemplary heterobifunctional linker in this regard has a maleimidereactive group at one end and a hydrazide reactive group at the otherend. One example of a heterobifunctional linker useful in this regardis: ##STR5## which may be obtained by the following reaction scheme:##STR6##

Alternatively, the maleimide group of the described heterobifunctionallinker may be reacted with native sulfhydryl groups on the targetingprotein. Native sulhydryls may be generated from targeting proteindisulfide bonds through exposure of the targeting protein to a reducingagent, such as dithiothreitol.

The free hydrazide group of the targeting protein-linker molecule maythen be reacted with aldehyde or ketone groups of a diagnostic ortherapeutic agent. One technique thorough which aldehyde or ketonegroups may be generated on a diagnostic/therapeutic agent is byoxidation of oligosaccharides (in the case of a glycoprotein agent).With certain diagnostic/therapeutic agents, oxidation of the agent mayprovide additional in vivo benefits. For instance, oxidation of ricin Achain decreases delivery of ricin A to the mammalian liver.

A method for generating aldehyde/ketone groups on anon-carbohydrate-containing agent involves oxidation of methyl groups orprimary hydroxyl groups to form aldehydes, or oxidation of secondaryhydroxyl groups to form ketones. For example, the secondary hydroxylgroup at the 2' position of the macrocyclic ring of verrucarin A may beoxidized to a ketone. Reaction of the targeting protein-linker andoxidized agent may be represented by the following scheme: ##STR7##

Alternatively, an aldehyde group may be introduced into anon-carbohydrate-containing diagnostic or therapeutic agent through useof a heterobifunctional linker having a free reactive aldehyde at oneend. An example of a heterobifunctional linker useful in this regard is:##STR8## which may be obtained b he following reaction scheme: ##STR9##

The maleimide group of this heterobifunctional linker reacts with nativesulfhydryl groups on the diagnostic or therapeutic agent (generatedthrough treatment with a reducing agent) or with asulfhydryl-derivatized agent (iminothiolane-generated sulfhydryls fromnative lysines of the agent).

The free hydrazide group of the targeting protein-linker molecule isthen reacted with the aldehyde group of the agent-linker molecule,forming a stabilized unique Schiff base-linked targeting proteinconjugate. This reaction is schematically illustrated below: ##STR10##

Another heterobifunctional linker useful in this regard has a freereactive aldehyde at one end and an N-hydroxysuccinimide ester at theother end. An exemplary linker in this regard includes: ##STR11##

Lysines of the diagnostic or therapeutic agent react with theN-hydroxysuccinimide ester of the linker molecule. The free hydrazidegroup of the targeting protein is then reacted with the aldehyde groupof the agent-linker molecule, as described above.

In a second embodiment of the present invention, a targeting protein isreacted with a heterobifunctional linker having a maleimide reactivegroup at one end and an aldehyde reactive group at the other end. Anexample of a heterobifunctional linker useful in this regard has beenprovided above. The maleimide group of the linker reacts with sulfhydrylgroups of a targeting protein (generated as described above).

A diagnostic or therapeutic agent is converted into an agent-hydrazidethrough reaction with a maleimidehydrazide heterobifunctional linker (asdescribed previously). The free aldehyde group of the targetingprotein-linker is then reacted with the diagnostic/therapeuticagent-linker hydrazide, yielding a targeting proteinconjugate.,according to the following scheme: ##STR12##

In a third embodiment of the first aspect of the present invention,carboxylic acid groups of a targeting protein are directly derivatizedwith hydrazine in the presence of carbodiimide to form a targetingprotein hydrazide. The targeting protein hydrazide is then covalentlyattached through a stabilized unique Schiff base linkage to an aldehydeor ketone group present on a diagnostic or therapeutic agent. Thefollowing reaction scheme illustrates production of the resultantstabilized unique Schiff base-linked targeting protein conjugate:##STR13##

A variety of carbodiimides may be used as catalysts in the abovereaction scheme, but 1-ethyl-3(3-dimethylaminopropyl)carbodiimide is aparticularly preferred carbodiimide.

As depicted in the above reaction scheme, an aldehyde group may beintroduced into either a carbohydrate- or non-carbohydrate-containingdiagnostic: or therapeutic agent through use of a heterobifunctionallinker having a free reactive aldehyde at one end. In this illustrativeexample, L2 is present (n=1). Alternatively, aldehyde or ketone group(s)on the diagnostic/therapeutic agent may be obtained directly byoxidation of oligosaccharides (in the case of a glycoprotein agent) oroxidation of methyl or secondary hydroxyl groups. When oxidation is usedto generate aldehyde/ketone groups on the agent, L2 is not present(n=0).

Crosslinking of targeting protein during reaction with hydrazine isminimized by maintaining the concentration of targeting protein atapproximately 2 mg/ml, and by using an excess of hydrazine reactant.

A comparison of the Schiff base-linked conjugates of the first aspect ofthe claimed invention and known Schiff base-linked conjugates highlightsthe following advantages provided by the conjugates described herein:(1) Neither the targeting protein nor the diagnostic or therapeuticagent need be a glycoprotein. (2) If the targeting protein and/or thediagnostic or therapeutic agent component of the conjugate is aglycoprotein, this glycoprotein component need not be subjected to harshoxidizing conditions in order to generate a Schiff base-linkedconjugate. This is in contrast to previously described Schiffbase-linked conjugates, which require generation of oxidizedcarbohydrate moieties. (3) Either sulfhydryl, ε-amino or carboxylic acidgroups of the targeting protein may be derivatized to in readiness forunique Schiff base-linkage of the agent. (4) In contrast to previouslydescribed Schiff base-linked conjugates which require oxidizedoligosaccharide moieties, the degree of conjugation of the targetingprotein or diagnostic or therapeutic agent of the present invention maybe controlled (for instance, through the amount of hydrazide or linkersubstituted onto the targeting protein and/or agent components). WhereSchiff base-linked conjugates are obtained using oxidizedoligosaccharides, the degree of conjugation is directly related to theamount of carbohydrate natively associated with the glycoprotein. (5)Stabilized Schiff base conjugates of the claimed invention need not besubjected to reducing conditions in order to stabilize a Schiff base(imine) bond.

A second aspect of the present invention involves a targetingprotein--diagnostic/therapeutic agent conjugate joined through aheterobifunctional, aromatic Schiff base linker. In a first embodimentof this aspect of the invention, a targeting protein conjugate has thefollowing formula: ##STR14## wherein "Ar" is a substituted orunsubstituted aryl group derived from an aromatic aldehyde or ketonehaving the formula R--CO--Ar;

"N" is a nitrogen atom contributed by the targeting protein;

"C" is a carbon atom contributed by the aromatic aldehyde or ketone;

"R" is H or an alkyl, aryl or heteroaryl substituent contributed by thearomatic aldehyde or ketone; and

"Z" is a diagnostic or therapeutic agent attached either directly orindirectly to Ar.

Preferred Ar groups in this regard include monocyclic aromatic rings,annulated aromatic rings, carbocyclic aromatic rings and heterocyclicring systems. Particularly preferred Ar groups include substituted andunsubstituted benzene, furan, pyrrole, thiophene, pyridine, oxazole,imidazole, thiazole and annulated derivatives thereof. A preferredannulatad Ar contains 2 to 5 rings.

According to this aspect of the claimed invention, aromatic aldehydes oraromatic ketones are designed and synthesized to act as reversible, acidcleavable linkers useful for controlled release of a diagnostic ortherapeutic agent from a targeting protein-diagnostic/therapeutic agentconjugate. It is contemplated that the stability of the Schiff base.(imine) linkage depicted above may be modified by altering theelectron-withdrawing or electron-donating nature of the linker aromaticring.

Previously, procedures for Schiff base conjugation of a protein and acarbonyl compound required a final reductive step (reductive amination)for stabilization of the resultant imine linkage. Alternatively, in thecase of α-hydroxy aldehydes (such as glyceraldehyde) or glucose, theSchiff base would undergo rearrangement to achieve a more stable product(Amadori rearrangement).

In contrast to previously described Schiff base linkages between proteinand an aldehyde or ketone compound, the claimed targeting proteinconjugate does not require stabilization of the Schiff base linkage(either through reduction or rearrangement). Instead, unreduced iminebond stability is achieved by altering the electron-withdrawing orelectron-donating characteristics of the linker aromatic ringsubstituents. For instance, substitution of ortho-hydroxy groups and/orelectron-donating groups on the aromatic ring of the Schiff base linkerwould increase the lability of the imine bond linkage to acidicconditions. Electron-withdrawing substituents on the aromatic ring ofthe Schiff base linker would stabilize the imine bond linkage to acidicconditions.

Preferred electron-donating groups in this regard include O⁻, S⁻, NR'₂,NHR', NH₂, NHCOR', OR', OH, OCOR', SR', SH, Br, I, Cl, F and R'.Preferred electron-withdrawing groups in this regard include NO₂, CN,CO₂ H, CO₂ R', CONH₂, CONHR', CONR'₂, CHO, COR', SO₂ R', SO₂ OR' and NO.Within the electron-donating and electron-withdrawing groups, R' may beH; a substituted or unsubstituted alkyl, aryl or heteroaryl group; asubstituent that increases water solubility of the linker; or asubstituent that further affects the stability of the resultant Schiffbase linkage.

Within the second aspect of the invention, "Z" indicates a diagnostic ortherapeutic agent that is either directly or indirectly attached to Ar.Preferred Z substituents include a directly-linked radionuclide; afunctional group suitable for linking a cytotoxic agent; a chelatingligand capable of binding a radiometal; and an organometallicsubstituent, such as aryltin, that is susceptible to replacement by aradiohalogen.

The Ar group of the linker may be derivatized with a Z substituent priorto covalent linkage of a targeting protein amine group and an aldehydeor ketone moiety present on Ar (generating a unique Schiff baselinkage). If Z is a functional group suitable for linking a cytotoxicagent, a chelating ligand capable of binding a radiometal, or anorganometallic substituent, the Z substituent may be reacted with acytotoxic agent, a radiometal or a radiohalogen subsequent toconjugation of Ar--Z and a targeting protein.

Alternatively, the Z substituent of the aromatic linker of the secondaspect of the invention may be first reacted with a cytotoxic agent, aradiometal or a radiohalogen, thereby forming an R--CO--Ar--Z!-diagnostic/therapeutic agent compound. " Z!" indicates that theprereaction Z substituent may or may not remain after reaction of Z withthe diagnostic or therapeutic agent. The R--CO--Ar--Z!-diagnostic/therapeutic agent compound is then conjugated with atargeting protein via a Schiff base linkage formed between the R--CO--Argroup of the linker and a targeting protein amine group.

Preferred Z functional groups include activated esters (which react withamino groups), maleimides (which bind to sulfhydryl groups) andhaloacetamides (which also bind to sulfhydryl groups). In a particularlypreferred embodiment, Z is an N-hydroxysuccinimide ester, whichpossesses electron-withdrawing properties that increase the acidstability of the Schiff base linkage between the targeting protein andthe Ar group. In another particularly preferred embodiment, Z is abromoacetamide group, which has electron-donating characteristics thatdecrease the acid stability of the Schiff base linkage of the conjugate.

Exemplary bifunctional linkers and their corresponding synthetic routesare shown below:

    __________________________________________________________________________    Formula:                                                                      __________________________________________________________________________     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                    __________________________________________________________________________

Preferred Z chelating ligands include radionuclide metal chelates asdescribed in Fritzberg, EP 188,256. Particularly preferred chelates inthis regard have a free NH₂ group capable of reaction with Z when Z isan activated ester or have an available maleimide group capable ofreaction with Z when Z is a sulfhydryl. Yet another preferred Zchelating ligand is an aryltin group, as described in Wilbur et al, EP203,764, with tributyltin particularly preferred. Radiohalogens may thenbe attached to linker substituent Ar by halo-destannylation.

Z may also represent a radionuclide directly attached to the Ar linkersubstituent, with ¹²⁵ I p-iodobenzaldehyde a particularly preferredAr--Z compound. Appropriately substituted aromatic compounds that havebeen directly radioiodinated by addition of electrophilic iodine arealso preferred Ar--Z compounds. For instance, the hydroxy group ofo-vanillin activates an Ar ring for electrophilic attack by aradiohalogen (i.e., iodine). O-vanillin offers the further advantage ofregioselective radiohalogenation, since both Ar ring positions ortho tothe activating hydroxy group are occupied (see formula below). ##STR18##

Preferred diagnostic and therapeutic radionuclides that may be eitherdirectly or indirectly attached to Ar include gamma-emitters,positron-emitters, Auger electron-emitters, X-ray emitters andfluorescence-emitters, with beta- or alpha-emitters preferredtherapeutic agents. Radionuclides are well-known in the art and include¹²³ I, ¹²⁵ I, ¹³⁰ I, ¹³¹ I, ¹³³ I, 135I, ⁴⁷ Sc, ⁷² As, ⁷² Se, ⁹⁰ Y, ⁸⁸Y, ⁹⁷ Ru, ¹⁰⁰ Pd, ^(101m) Rh, ¹¹⁹ Sb, 128Ba, ¹⁹⁷ Hg, ²¹¹ At, ²¹² Bi, ²¹²Pb, ¹⁰⁹ Pd, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁷ Cu, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^(99m) Tc,¹¹ C, ¹³ N, ¹⁵ O and ¹⁸ F. Preferred therapeutic radionuclides include¹⁸⁸ Re, ¹⁸⁶ Re, ²⁰³ Pb, ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵I, ¹³¹ I, ⁷⁷ Br, ²¹¹ At, ⁹⁷ Ru, 105Rh, ¹⁹⁸ Au and ¹⁹⁹ Ag.

Intracellular release of a cytotoxic agent, a chelating ligand plusagent or a radionuclide from a targeting protein conjugate may bedesirable in many instances. In this regard, the claimed diagnostic ortherapeutic targeting protein conjugate provides serum stability duringdelivery of the conjugate to an appropriate target cell. Uponinternalization of the conjugate into target cell endosomes, theattached cytotoxic agent or radionuclide is released, which in turn mayfacilitate translocation of the diagnostic or therapeutic agent orradionuclide from the target cell endosome into the cytoplasm. In thecase of certain proteinaceous agents, translocation into the cytoplasmwould allow the agent to escape degradation in target: cell lysosomes.

Some proportion of administered targeting protein conjugate will bind tonormal cells of the mammalian recipient. Typically, if antibody isconjugated to a radiometal using non-cleavable bifunctional linkers,accumulation of significant amounts of radionuclide in normal tissues(i.e., liver and bone marrow) by receptor-mediated endocytosis isobserved.

In contrast, the conjugate of the second aspect of the inventionprovides a reversible (acid-cleavable) attachment of thediagnostic/therapeutic agent to a targeting protein. The claimedconjugate might provide reduced accumulation of the radionuclide innormal tissues, through release of the covalently attached radiometalinto the acidic environment of the normal cell endosome/lysosome. As aresult, the diagnostic/therapeutic agent (with or without chelator) maybe subject to accelerated metabolism and excretion by the normal cell.When the agent is shunted cut of the normal cell, it is returned to thebloodstream and rapidly excreted by the kidney, rather than accumulatingin normal tissues.

In a second embodiment of the second aspect of the claimed invention, atargeting protein--diagnostic/therapeutic agent conjugate joined by aSchiff base linkage may be synthesized according to the followingreaction scheme: ##STR19## wherein "X" is (CH₂)_(n) --Y, where n=0-6,Y=active ester, isothiocyanate or maleimide, and X is substituted at the3 and/or 5 position of Ar;

"Ar" is aryl substituted with electron-donating and/orelectron-withdrawing groups at the 2, 4 and/or 6 position;

"R" is H or an alkyl, aryl or heteroaryl substituent contributed by thearomatic aldehyde or ketone; and

"agent" is an amino-containing diagnostic or therapeutic agent; .namino-containing chelator for a diagnostic or therapeutic agent; or anamino-containing aromatic organometallic.

In this second embodiment the resultant Schiff base linkage is formedbetween a diagnostic or therapeutic agent amine and aheterobifunctional, aromatic linker. This is in contrast to the Schiffbase linkage in the first embodiment, which is formed between atargeting protein amine and a heterobifunctional, aromatic linker.

The effect of Ar substituents on the formation of Schiff base linkagesand on conjugation to a targeting protein should be negligible. However,the rate of hydrolysis of the diagnostic or therapeutic agent from thetargeting protein conjugate may be influenced by Ar substituents. Forinstance, the presence of electron-donating groups at positions 2, 4,and/or 6 of Ar will enhance release of the agent from the conjugateunder acidic conditions; the presence of electron-withdrawing groups atpositions 2, 4 and/or 6 of Ar will retard or inhibit the release of theagent from the targeting protein conjugate.

Preferred electron-donating groups in this regard include O⁻, S⁻,^(NR'2), NHR', NH₂, NHCOR', OR', OH, OCOR', SR', SH, Br, I, Cl, F andR'. Preferred electron-withdrawing groups in this regard include NO₂,CN, CO₂ H, CO₂ R', CONH₂, CONHR', CONR'₂, CHO, COR', SO₂ R', SO₂ OR' andNO. Within the electron-donating and electron-withdrawing groups, R' maybe H; a substituted or unsubstituted alkyl, aryl or heteroaryl group; asubstituent that increases water solubility of the linker; or asubstituent that further enhances the stability of the resultant Schiffbase linkage.

In this embodiment, the targeting protein conjugate is serum stable,but, upon binding at a target cell surface, may be susceptible to thioladdition to the imine (C═N) bond. Thiol addition to the C═N bond of theconjugate would result in hemithioaminal formation (depicted below).##STR20## Any effect of Ar substituents on the reaction of the conjugateimine bond with cell surface sulfhydryl groups would be negligible.

Optimal in vivo diagnostic or therapeutic efficacy of targeting proteinconjugates may involve three levels of conjugate-target cell membraneinteraction: (1) binding of the conjugate to the cell surface membrane;(2) internalization of the conjugate into target cell endosomicvesicles; and (3) translocation of the conjugate from endosomic vesiclesinto the target cell cytoplasm.

Upon administration of the targeting protein conjugate of thisembodiment of the second aspect of the invention, formation of ahemithioaminal would increase retention of the targeting proteinconjugate at the cell surface through covalent attachment. Increasedretention of the conjugate thus may result in increased internalizationand translocation of the conjugate, which in turn may increase theefficacy certain diagnostic/therapeutic agents. For instance,translocation of the conjugate is particularly desirable if thecytotoxic agent component acts on a cytoplasmic target.

To summarize the examples that follow, Example I describes formation ofa stabilized unique Schiff base targeting protein (monoclonal antibody)conjugate; Example II discusses a stabilized unique Schiff baseconjugate of human serum albumin and 16-oxo-verrucarin A. Synthesis of atargeting protein conjugate joined through an ¹²⁵ I benzaldehydederivative linker is shown in Example III. Production of a targetingprotein conjugate joined, through a substituted acetophenone linker isdescribed in Example IV.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLE I Formation of Stabilized Unique Schiff Base Targeting ProteinConjugate A. Using Targeting Protein Sulfhydryls

Formation of a targeting protein hydrazide is achieved by treatingmonoclonal antibody (MAb; 5 mg/ml in phosphate-buffered saline PBS!, pH8.5) with 10 mM dithiothreitol (DTT). The reaction mixture is agitatedat room temperature for 30 minutes, and the reduced MAb is passedthrough a PD-10 column (Pharmacia, Uppsala, Sweden) to remove unreactedDTT.

The reduced monoclonal antibody is derivatized with a heterobifunctionallinker of Formula 1. Any targeting protein containing one or more nativedisulfide bonds can thus be converted to a targeting protein hydrazide.

The trichothecene therapeutic agent 16-oxo verrucarin A is prepared byselenium dioxide oxidation of verrucarin A. The MAb hydrazide is thenreacted with 16-oxo verrucarin A at 4° C. overnight with agitation,thereby yielding a unique Schiff base-linked targeting protein, havingthe following structure: ##STR21##

B. Using Targeting Protein Lysines

Monoclonal antibody (5 mg/ml in PBS, pH 8.5) is treated withiminothiolane (IT); the amount of IT offered to the MAb preparation willbe dependent on the number of free MAb sulhydryls desired. The reactionof IT with targeting protein lysines is schematically represented asfollows: ##STR22## The reaction mixture is agitated at room temperaturefor 30 minutes, then passed through a PD-10 column to remove unreactedIT. The sulfhydryl-derivatized MAb is then reacted with aheterobifunctional linker of Formula 1 to form a MAb hydrazide, asdescribed in Example I.A.

Ricin A chain is oxidized with 10 mM NaIO₄, pH 5.5 at room temperaturefor 1 hour to generate free aldehyde groups from native ricin Aoligosaccharide moieties. A significant advantage obtained throughunique Schiff base linkage of ricin A aldehyde groups is that oxidationof ricin A decreases the amount of ricin A non-specifically delivered tothe mammalian liver (as compared with non-oxidized ricin A).

The oxidized ricin A is then reacted with the MAb hydrazide producedabove, yielding a unique Schiff base targeting protein having thefollowing formula: ##STR23##

Iminothiolane-derivatization may be used to generate free sulfhydryls onany lysine-containing protein or peptide. A heterobifunctional linkerhaving a reactive maleimide on one end and a hydrazide group on theother end may then be used to form a proteinaceous hydrazide from alysine-containing protein or peptide (i.e., regardless of whether theprotein or peptide contains native disulfide bonds).

C. Using Targeting Protein Carboxylic Acid Groups

A solution of monoclonal antibody (2 mg/ml in PBS, pH 6.5) is reactedwith excess hydrazine (1:150) and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (1:100) and agitated forapproximately 1 hour at room temperature to form a hydrazide of the MAb.Reaction conditions (reactant ratios and reaction time) may varysomewhat depending on the monoclonal antibody used. At the end of the 1hour incubation, the monoclonal antibody hydrazide is passed through aPD-10 column to remove unreacted hydrazine and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide.

The reaction described directly introduces hydrazine onto the MAb,without the use of a heterobifunctional linker having a reactivehydrazine group. Dimerization (crosslinking) of MAb is prevented bymaintaining a concentration of targeting protein (MAb) of approximately2 mg/ml, and by using a large excess of hydrazine reactant. Further, thereaction described may be used with any targeting protein that containsone or more free carboxylic acid groups, whether glycoprotein ornon-glycoprotein.

EXAMPLE II Formation of Stabilized Unique Schiff Base Conjugate of HumanSerum Albumin and Verrucarin A

A solution of human serum albumin (HSA) is reacted with1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide as in Example I.C. toform a hydrazide of HSA. These reaction conditions favor formation ofthe hydrazide with minimization of HSA crosslinking. The HSA hydrazideis then reacted with 16-oxo-verrucarin A, as described in Example I, toform a conjugate of HSA and 16-oxo-verrucarin A that is joined by astabilized unique Schiff base linkage.

EXAMPLE III Synthesis of a Targeting Protein Conjugate Joined Through a¹²⁵ I Benzaldehyde Derivative Linker

Radiolabeled benzaldehyde linkers demonstrate the relationship betweenaromatic aldehyde ring substituents and acid-catalyzed hydrolysis ofSchiff base-containing-radionuclide-targeting protein conjugates.

Briefly, ¹²⁵ I-p-iodobenzaldehyde was prepared as follows: ##STR24## Thetri-n-butyltin precursor to p-iodobenzaldehyde was prepared fromcommercially available p-bromobenzyl alcohol (Aldrich Chemical Co.,Milwaukee, Wis.). Treatment of the p-bromobenzyl alcohol compound withhexabutylditin and Pd(PPh₃)₄ in toluene yielded the desired aryl tincompound. The alcohol was oxidized to the corresponding aldehyde withpyridinium chlorochromate (PCC) in methylene chloride solvent. Theproduct was isolated in 69% yield after silica gel chromatography (ethylacetate/hexanes) as a pale yellow oil. ¹ H NMR (CDCl₃) δ 0.83-1.61 (m,27H); 7.64 (d, J=8Hz, 2H); 7.78 (d, J=8 Hz, 2Hz); 9.98 (s, 1H); ¹³ C NMR(CDCl₃) δ9.88, 13.82, 27.54, 29.25, 128.99, 129.59, 136.97, 137.50,193.61.

A trace radiolabeling was accomplished with NCS/¹²⁵ I to give thedesired product, as evidenced by coinjection with a cold standard. Thereaction conditions for labeling were as follows: To a solution of 0.05mg of the benzaldehyde derivative in 0.05 ml of 1% acetic acid/MeOH wasadded approximately 360 μCi Na¹²⁵ I and 0.0019 mg NaI carrier (1 mg/mlMeOH). To this mixture was added 17 μl of NCS (1 mg/ml in MeOH). After30 minutes at room temperature, 17 μl of sodium bisulfite solution (0.72mg/ml in water) was added to quench the reaction. The radiochemicalyield obtained was 93%.

Upon conjugation of ¹²⁵ I-p-iodobenzaldehyde to monoclonal antibodyNR-ML-05 (11:1 aldehyde:protein) at pH 9.0, 58% of the offeredradiolabeled aldehyde was covalently attached to antibody.

The radionuclide-monoclonal antibody conjugate was purified bycentrifugation (5,000 RPM, 10 min, room temperature) through a 30,000 MWmicroconcentrator (Amicon, Danvers, Mass.), yielding a conjugate of 97%purity by instant thin layer chromatography (ITLC). An analysis ofspecific activity of the purified conjugate indicated that approximatelytwo lysine residues per antibody molecule had been modified.

Radiolabeled 5-iodo-3-methoxysalicylaldehyde and5-iodo-3-methylsalicylaldehyde (below) are prepared by radioiodinationof o-vanillin and 3-methylsalicylaldehyde, respectively. ##STR25##Radioiodination of o-vanillin and 3-methylsalicylaldehyde should beregioselective, since both positions ortho to the hydroxy substituent ofthe aromatic ring are occupied.

The iodinated aromatic aldehydes illustrated above should demonstratedecreased acid stability as compared to ¹²⁵ I-p-iodobenzaldehyde, sincethe ortho-hydroxy substituents can protonate a Schiff base linkage viaan intramolecular mechanism. More particularly, the meta methoxy groupof 5-iodo-3-methoxysalicylaldehyde has slight electron-withdrawingproperties that may increase the acid stability of the Schiff baselinkage; the meta methyl group of 3-methylsalicylaldehyde haselectron-donating characteristics, which should decrease the stabilityof the Schiff-base-linked conjugate. Conjugates containing radiolabeled5-iodo-3-methoxysalicylaldehyde and 5-iodo-3-methylsalicylaldehyde canbe characterized as having "stable" conventional Schiff base linkagesthat are cleavable under mildly acidic conditions (pH 5-6).

EXAMPLE IV Schiff Base-Linked Targeting Protein Conjugates Joined by aSubstituted Acetophenone Linker

A stable Schiff base linkage between an amine and an aromatic carbonylcompound is formed using adriamycin and substituted or unsubstitutedacetophenone, as shown below: ##STR26## Briefly, formation of a Schiffbase linkage is accomplished by briefly refluxing equimolar amounts ofadriamycin and substituted acetophenone, or by mixing these reactants inan aprotic solvent in the presence of a dehydrating agent (for instance,a molecular sieve). If the acetophenone is substituted with a --COOHgroup, the --COOH is converted to an active ester after formation of theSchiff base-linked adriamycin-acetophenone compound. Alternatively, ifthe acetophenone is substituted with a maleimide group, no furtherreaction of the acetophenone prior to reaction with targeting protein isnecessary.

The Schiff base-linked adriamycin-acetophenone compound is conjugatedwith monoclonal antibody (MAb) (i.e., intact MAb, F(ab')₂ fragment,F(ab') fragment or Fab fragment). If the targeting protein-reactivegroup of the adriamycin-acetophenone compound is an active ester, thereaction with MAb will preferably be done at pH 8-10. If the targetingprotein reactive group of the adriamycin-acetophenone compound ismaleimide, the reaction with MAb will preferably be done at pH 6-7. Arange of concentrations of adriamycin-acetophenone compound and MAb willbe tested in order to determine what concentration of each yieldsoptimal immunoreactivity of the resultant targeting protein conjugate.The optimal concentration of targeting protein reactant required toachieve maximum conjugate immunoreactivity will vary depending on theparticular targeting protein to be conjugated (i.e., antibody vs.hormone; one MAb vs. another MAb). The resultant targetingprotein-therapeutic agent conjugate may be depicted as follows:##STR27##

A therapeutically effective amount of the monoclonal antibody-adriamycinSchiff base-linked conjugate is administered intravenously to atumor-bearing patient. Upon binding of the targeting protein-drugconjugate to an appropriate tumor target cell, thiol groups present atthe tumor cell membrane surface may add to the C═N Schiff base iminebond, producing a hemithioaminal (as depicted below). ##STR28##Formation of the hemithioaminal increases retention of the monoclonalantibody-drug conjugate at the tumor cell surface, which in turn maylead to increased tumor cell cytotoxicity.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. An improved method of diagnosis or therapy whichcomprises administration of an antibody or antigen binding antibodyfragment targeting protein radionuclide conjugate wherein theimprovement comprises administering an antibody or antigen bindingantibody fragment conjugated to a radionuclide by a Schiff base linkerand having the formula:

    antibody or antigen binding antibody fragment ##STR29## wherein "Ar" is a substituted or unsubstituted aryl group;

"N" is a nitrogen atom; "C" is a carbon atom; "R" is H or an alkyl, arylor heteroaryl substituent; and "Z" comprises a radionuclide selectedfrom the group consisting of ¹²³ I, ¹²⁵ I, ¹³⁰ I, ¹³¹ I, ¹³³ I, ¹³⁵ I,⁴⁷ Sc, ⁷² Se, ⁹⁰ Y, ⁸⁸ Y, ⁹⁷ Ru, ¹¹⁹ Sb ,¹⁹⁷ Hg, ²¹¹ At, ²¹² Bi, ²¹² Pb,¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁷ Cu, ⁷⁵ Br, ⁷⁶ Br, ^(99m) Tc, ¹⁸ F, ¹⁸⁸ Re, ¹⁸⁶Re, ²⁰³ Pb, ⁶⁴ Cu, ¹⁰⁵ Rh, ¹⁹⁸ Au and ¹⁹⁹ Au; and wherein thesubstituent of Ar, if present, is an electron withdrawing group or anelectron-donating group capable of modifying unreduced imine bondstability of the Schiff base linker, and wherein the Schiff base isformed by a process which comprises: reacting a non-oxidized and nativeamine-bearing or amine-derivatized targeting protein with an aromaticaldehyde or ketone, wherein the aromatic aldehyde or ketone is directlyconjugated to Z or indirectly conjugated to Z via a chelating ligand, toform a stabilized Schiff base linker, absent a subsequent stabilizingstep with respect to the C═N bond; wherein antibody or antigen bindingantibody fragment carbohydrate content is not determinative of maximumpossible aromatic aldehyde or ketone conjugation to the antibody orantigen binding antibody fragment.
 2. The method of claim 1, wherein Aris selected from the group consisting of a monocyclic aromatic ring, anannulated aromatic ring, a carbocyclic aromatic ring and a heterocyclicring system.
 3. The method of claim 1, wherein Ar is selected from thegroup consisting of substituted and unsubstituted benzene, furan,pyrrole, thiophene, pyridine, oxazole, imidazole, thiazole and annulatedderivatives thereof.
 4. The method of claim 1, wherein theelectron-withdrawing group is selected from the group consisting of NO₂,CN, CO₂ R', CONH₂, CONHR', CONR'₂, CHO, COR', SO₂ R, SO₂ R' and NO,wherein R' may be H, a substituted or unsubstituted alkyl, aryl orheteroaryl, a substituent that increases water solubility, or asubstituent that affects Schiff base linkage stability.
 5. The method ofclaim 1, wherein the electron-donating group is selected from the groupconsisting of O⁻, S⁻, NR'₂, NHR', NH₂, NHOR', OR', OH, OCOR', SR', SH,Br, I, Cl, F and R', wherein R' may be H, a substituted or unsubstitutedalkyl, aryl or heteroaryl, a substituent that increases watersolubility, or a substituent that affects Schiff base linkage stability.6. The method of claim 1, wherein Z is a radiohalogen.
 7. The method ofclaim 1, wherein Z comprises ^(99m) Tc, ¹⁸⁸ Re, or ¹⁸⁶ Re bound by achelating ligand.